Injectable liposomal depots for delivering active ingredients

ABSTRACT

The invention relates to liposomal formulations for the production of an injectable depot of peptide, protein and oligonucleotide active substances for sustained release and effect in a mammal body.

The invention relates to a liposomal delivery system for the delayedrelease of active substances and to the use of said system in basicresearch and clinics.

Following application, peptide and protein active substances undergovery rapid degradation in the body or elimination and therefore must beadministered by repeated injections. To increase the “patientcompliance”, a suitable delivery system is required which protects theactive substance from degradation in the body, gradually releasing itinto the bloodstream. Depot systems being injected subcutaneously orintramuscularly or implanted are used to this end. Liposomes are onepossible form of such a carrier system. They are constituted of one ormore lipid double layers that enclose in their inside an aqueouscompartment allowing entrapment of water-soluble substances. The lipiddouble layer allows incorporation of lipophilic substances.

J. Controll. Rel. 64 (2000), 155-166, U.S. Pat. No. 5,766,627 and otherpapers by the authors present multivesicular aggregates of liposomes asinjectable depot system for insulin, leuprolides and enkephalin, whichare obtained by means of a double-emulsion process. Due to the additionof non-polar triglycerides, these multi-centered aggregates cannot beregarded as liposomes in a stricter sense because the triglycerides donot form any bilayer membranes and are not incorporated in the latter.Another drawback is that a water-immiscible oil phase is used in theproduction of said structures. Inclusion of larger proteins, inparticular, will give rise to denaturation at the interface. Likewise,residues of organic solvents represent a regulatory problem that shouldnot be underestimated.

According to the state of the art, liposomes composed of neutral,anionic or PEG lipids are used for depot systems, e.g. in WO 9920301 fora depot of γ-interferon, in Diabetes 31 (1982), 506-511, for a depot ofinsulin; furthermore, in Proc. Natl. Acad. Sci. 88 (1991), 10440-10444for vaccination.

In BBA 1328 (1997), 261-272, various liposomal systems (unilamellar andmultilamellar) of egg PC, egg PG, DPPC, DPPG, HP and cholesterol havebeen investigated for their reception in the lymphatic system and theirbiodistribution following subcutaneous administration. The reviewarticle Advanced Drug Delivery Reviews 50 (2001), 143-156, represents acontinuation of the above investigations, demonstrating that liposomessmaller in size (<150 nm) migrate from a subcutaneous depot into thelymph.

According to the state of the art, neutral and negatively chargedliposomes have been used in liposomal depot systems. For migration intothe lymph to be absent, the liposomes must have a minimum size.

However, the production of large liposomes significantly greater than150 nm is associated with technical and regulatory problems. Morespecifically, desirable sterile filtration of the particles subsequentto the production thereof is no longer possible.

Apart from peptides and proteins, oligonucleotides are likewise degradedvery rapidly in the body by enzymes. In general, these active substancesare administered at high doses by intravenous injections which, however,must be repeated frequently. For improved “patient compliance” and toallow reduction of the dose, a suitable delivery system is thereforerequired which protects the active substance against degradation in thebody and effects slow and delayed liberation thereof.

Usually, delivery systems supporting the intracellular delivery ofactive substances following administration are in use today. Theseinclude liposomal systems, polymer-based systems (e.g. PEI) and viralcarriers. Such intracellular strategies of delivery can result in dosereduction of the active substances. However, reduction of the injectionscannot be achieved.

Another way of administering oligonucleotides involves depot systemsbeing applied locally and liberating the active substances uniformlyover a defined period of time. Such strategies of delivery do notnecessarily support intracellular delivery of the active substances;rather, they result in a steady-state level of the active substance inblood or tissue for that period of time. In this way, the injectionfrequency can be reduced and, in addition, dose reduction is possible asa result of maintaining the concentration of active substance.

Micro- or nanoparticles made of biocompatible polymers represent onepossible form of such a depot system. U.S. Pat. No. 6,555,525 describesthe delayed release of antisense oligonucleotides from PLGAmicrocapsules following subcutaneous injection in a mouse leukemiamodel. Delayed release of oligonucleotides from PLGA-based micro- ornanocapsules has also been described in numerous other publications (forexample, J. Drug Target. 5(4), 291-302, (1998); Gene Ther. 9 (23),1607-16, (2002); Antisense Nucleic Acid Drug Dev. 9(5), 451-8, (1999);J. Control. Release 37, 173-183, (1995)).

Other polymer-based systems for the delivery of nucleic acids have beendescribed in other printed documents. The authors of Methods: ACompanion to Methods in Enzymology 18, 286-295, (1999), suggest e.g. thepossible use of poly(hexyl cyanoacrylate) nanoparticles describedtherein as a depot system for oligonucleotides.

One drawback of micro- or nanoparticles made of polymers is theproduction process thereof. In most of such cases, emulsion processesmust be employed, using organic water-immiscible solvents. Thesesolvents must be completely removed after the end of the process. As aresult, they represent a regulatory problem that should not beunderestimated. Moreover, hydrolysis of the PLGA capsules gives rise tovery low pH values inside the capsules, thus possibly impairing theintegrity of the entrapped active substances. Thus, it is a well-knownfact that purine bases are removed by hydrolysis from the nucleic acidbackbone at low pH values.

Liposomes are another possible form of a carrier system foroligonucleotides. Numerous publications deal with the use of—mostlycationic—liposomal systems for the in vivo delivery of oligonucleotides(for example, Molecular Membrane Biology, 16, 129-140, (1999); BBA 1464,251-261, (2000); Reviews in Biology and Biotechnology, 1(2), 27-33,(2001)). However, all these systems involve the common fact that thelipid mixtures used are constituted of unsaturated lipids such as DOTAPor DOPE and for this reason lack serum stability. As a result, suchliposomes will rapidly release the enclosed active substance afterinjection. Also, complexes of preformed liposomes and nucleic acids(e.g. Lipoplexe) are frequently produced for the applications mentionedabove. As a consequence of such complex formation, or of liposomalformulations mostly unstable in serum, stability of the oligonucleotidesfor a prolonged period of time, as required for a depot, cannot beguaranteed.

The object of the invention was therefore to provide new stableliposomal depot formulations for protein and peptide active substancesand oligonucleotides, which would achieve long-term release of an activesubstance for at least one week and have good tolerability in anorganism. Another object was to provide depot systems which avoid “burstrelease” of active substance or, if therapeutically indicated, achieverapid initial partial release of active substance, followed by asustained release of active substance.

The above technical object is accomplished by means of a depot system,particularly for delayed release of active substances, said systemcomprising liposomes (a) with saturated synthetic phosphatidyl cholinesselected from the group of DMPC, DPPC and/or DSPC, (b) cholesterol witha percentage of from 35 to 50 mole-%, (c) cationic lipids selected fromthe group of DC-Chol, DAC-Chol, DMTAP, DPTAP and/or DOTAP with apercentage of from 5 to 20 mole-% in the liposomal membrane, and (d) aprotein and/or peptide active substance, said formulation of activesubstances in liposomes being present in the form of aggregates whenused as a depot. In addition to neutral lipids, such liposomespreferably comprise cationic lipids.

For example, positively charged liposomes undergo good aggregation withcomponents of the serum or interstitial fluid, remaining at the puncturepoint in this condition. Advantageously, diffusion of the depot awayfrom the puncture point is thus avoided.

The depots can be such in nature to either allow or prevent burstrelease. Depots with no burst release can be such that active substanceadhering on the outside of the liposomes is detached and removed. Whereburst release is advantageous, the active substance adhering on theoutside of the liposomes will not be detached and removed.

Various methods of entrapping the—especially water-soluble—activesubstance in liposomes of the depot system are known to those skilled inthe art. For inclusion of a desired active substance in liposomes, theactive substance is dissolved in a buffer solution, for example, whichis subsequently used to produce the liposomes. In the so-called passiveinclusion, the relative volume enclosed by the liposomes being formed isan important issue. In passive inclusion, the inclusion efficiency isincreased with increasing lipid concentration because the liquid volumeenclosed by the lipid double layer is increased.

The teaching according to the present application has a number ofadvantages. Neutral/negatively charged liposomes, or micro- andnanoparticles of polymers are known to date, which have been used forthe objects mentioned above.

The liposomes of the invention undergo aggregation with serum componentsand interstitial fluid components so that the depot remains at the siteof puncture, thus preventing e.g. migration into the lymph. The lipidcomposition of the invention includes saturated backbone lipidsproviding integrity of the liposomes even in the aggregated state andthus improved protection of the active substance or longer depot times.The production process performs without organic, water-immisciblesolvents possibly causing regulatory problems because complete removalthereof is difficult or damage to the active substance (proteins) mayoccur. There are no degradation products, as is the case with micro- andnanoparticles of polymers, which might do damage to the active substance(acid reaction during degradation of PLGA capsules). Depending on therequirements of therapy and on the active substance, variability isprovided by the present/absent burst release character of the depotsystem.

In a preferred embodiment of the present invention, liposomesconstituted of neutral and cationic lipids are used as liposomal depotsystem for the delayed release of therapeutic peptides and proteins of awide variety of molar masses. J. Pharm. Sci. 89(3), 297-310, 2000,describes the absolute bioavailabilities of peptides and proteins ofvarious size following subcutaneous application, wherein no significantreduction in bioavailability with increasing molar mass has beenobserved.

Therapeutic peptides and proteins undergo very rapid degradation in thebody, for which reason they must be administered by repeated injections.The peptides and proteins, analogs thereof, related peptides, fragments,inhibitors and antagonists relevant to this embodiment of the inventioncomprise:

Transforming growth factors (TGF-alpha, TGF-beta), interleukins (e.g.IL-1, IL-2, IL-3), interferons (IFN-alpha, IFN-beta, IFN-gamma),calcitonin, insulin-like growth factors (IGF-1, IGF-2), parathyroidhormone, granulocyte colony-stimulating factor (GCSF), granulocytemacrophage colony-stimulating factor (GMCSF), macrophagecolony-stimulating factor (MCSF), erythropoietin, insulins, amylins,glucagons, lipocortins, growth hormones, somatostatin, angiostatin,endostatin, octreotide, gonadotropin-releasing hormone (GNRH),luteinizing hormone-releasing hormone (LHRH), and effective agonistssuch as leuprolide acetate, buserelin, goserelin, triptorelin;platelet-derived growth factor; blood-clotting factors (e.g. factorVIII, factor IX), thromboplastin activators, tissue plasminogenactivators, streptokinase, vasopressin, muramyl dipeptides (MDP), atrialnatriuretic factor (ANF), calcitonin gene-related peptide (CGRP),bombesin, enkephalins, enfuvirtides, vasoactive intestinal peptide(VIP), epidermal growth factor (EGF), fibroblast growth factor (FGF),growth hormone-releasing hormone (GRH), bone morphogenetic proteins(BMP), antibodies and antibody fragments (e.g. scFv fragments, Fabfragments), peptide T and peptide T amides, herpes virus inhibitor,virus replication inhibition factor, antigens and antigen fragments,soluble CD4, ACTH and fragments, angiotensins, and ACE inhibitors,bradykinin (BK), hypercalcemia malignancy factor (PTH-like adenylatecyclase-stimulating protein), beta-casomorphins, chemotactic peptidesand inhibitors, corticotropin-releasing factor (CRF), caerulein,cholecystokinins+fragments and analogs, galanin, gastric inhibitorypolypeptide (GIP), gastrins, gastrin-releasing peptide (GRP), motilin,PHI peptides, PHM peptides, peptide YY, secretins,melanocyte-stimulating hormone (MSH), neuropeptide Y (NPY), neuromedins,neuropeptide K, neurotensins, phosphate acceptor peptide (c-AMP proteinkinase substrates), oxytocins, substance P, TRH, as well as fragments,analogs and derivatives of the above substances.

Another preferred class of active substances for liposomal depotsaccording to the invention are oligonucleotides. Oligonucleotidesrelevant to this embodiment of the invention are constituted of 5-100,preferably 5-40 and more preferably 10-25 nucleotides or base pairs.Moreover, the oligonucleotides can be present as a single strand (e.g.antisense oligonucleotides), double strand (e.g. small interfering RNA,decoy oligonucleotides), or in complex folding (e.g. aptamers,spiegelmers, ribozymes) . All oligonucleotides relevant to thisinvention are constituted of deoxyribonucleotides or ribonucleotides andchemically modified derivatives thereof (e.g. phosphorothioate DNA (PS),2′-O-methyl-RNA (OMe), 2′-O-methoxyethyl-RNA (MOE), peptide nucleic acid(PNA), N3′-P5′-phosphoroamidate (NP), 2-fluroarabino nucleic acid(FANA), locked nucleic acid (LNA), morpholinophosphoroamidate (MF),cyclohexene nucleic acid (CeNA), tricyclo-DNA (tcDNA)). Moreover,copolymers and block copolymers of various nucleotides and so-calledgapmers can be enclosed in the liposomes.

In one advantageous embodiment of the invention, aptamers or spiegelmersare enclosed in the liposomal depot. Aptamers are DNA- or RNA-basedoligonucleotides with a complex three-dimensional structure. Owing tothis structure, aptamers can bind to protein targets with highspecificity and high affinity, thus having a therapeutic, mostlyextracellular effect. Their functionality is virtually identical to thatof monoclonal antibodies.

Unlike D-oligonucleotides, spiegelmers are constituted of L-ribose andL-2′-deoxyribose units. Just like aptamers, these mirror image nucleicacids specifically bind to protein targets. Owing to the chiralinversion, spiegelmers—in contrast to conventionalD-oligonucleotides—have increased stability with respect to enzymaticdegradation.

Furthermore, water-soluble active substances or water-solublederivatives of active substances from the following classes of activesubstances are relevant to this invention: antibiotics (e.g. rifamycinSV Na salt, rifampicin, tetracyclin hydrochloride, kanamycin, penicillinG, ampicillin, novobiocin), antimycotic agents (e.g. amphotericin B,flucytosine), cytostatic agents (e.g. doxorubicin, daunorubicin,vincristin, cytarabin) , glucocorticoids (dexamethasone, prednisolone,hydrocortisone, betamethasone).

In addition to the above-mentioned classes of active substances,carbohydrates such as heparin or hyaluronic acid can be active substancemolecules relevant to this invention. Membrane proteins, being difficultto introduce in the inner space of liposomes, do not represent preferredactive substances in the meaning of the invention.

Membrane-forming and membranous lipids are possible as liposome-formingagents, and they can be of natural or synthetic origin. Morespecifically, these include cholesterol and derivatives, phosphatidylcholines, phosphatidyl ethanolamines as neutral lipids. In aparticularly preferred fashion, completely saturated compounds from thisclass are used, such as dimyristoyl, dipalmitoyl or distearoylderivatives of phosphatidyl cholines (DMPC, DPPC, DSPC) and phosphatidylethanolamines.

For example, cationic lipids used in the practice of the inventioncomprise:

-   -   DAC-Chol        3-β-[N-(N′,N′-dimethylaminoethane)carbamoyl]-cholesterol    -   DC-Chol        3-β-[N-(N′,N′-dimethylaminoethane)carbamoyl]-cholesterol,    -   TC-Chol        3-β-[N-(N′,N′,N′-trimethylaminoethane)carbamoyl]cholesterol,    -   BGSC Bis-guanidinium-spermidine-cholesterol,    -   BGTC Bis-guanidinium-tren-cholesterol,    -   DOTAP (1,2-dioleoyloxypropyl)-N,N,N-trimethylammonium chloride,    -   DOSPER (1,3-dioleoyloxy-2-(6-carboxyspermyl)propylamide),    -   DOTMA (1,2-dioleyloxypropyl)-N,N,N-trimethylammonium chloride        (Lipofectin®),    -   DORIE (1,2-dioleyloxypropyl)-3-dimethylhydroxyethylammonium        bromide,    -   DOSC (1,2-dioleoyl-3-succinyl-sn-glycero choline ester),    -   DOGSDSO (1,2-dioleoyl-sn-glycero-3-succinyl-2-hydroxyethyl        disulfide ornithine),    -   DDAB dimethyldioctadecylammonium bromide,    -   DOGS ((C18)₂GlySper3⁺) N,N-dioctadecylamido-glycyl-spermine        (Transfectam®),        (C18)₂Gly⁺N,N-dioctadecylamidoglycine, DOEPC        1,2-dioleoyl-sn-glycero-3-ethylphosphocholine or other        O-alkylphosphatidyl cholines or ethanolamines,        1,3-bis(1,2-bis-tetradecyloxy-propyl-3-dimethylethoxyammonium        bromide)-propan-2-ol (Neophectin®), and the saturated        derivatives with dimyristoyl, dipalmitoyl or distearoyl chains        of all above-mentioned lipids with unsaturated fatty acid and/or        fatty alcohol chains.

Preferred cationic lipids used in the practice of the invention comprisecholesteryl-3-β-N-(dimethylaminoethyl) carbamate (DC-Chol),3-β-[N-(N,N′-dimethylaminoethane)carbamoyl]cholesterol (DAC-Chol),(N-[1-(2,3-dimyristoyloxy)propyl]-N,N,N-trimethylammonium salt (DMTAP),(N-[1-(2,3-dipalmitoyloxy)propyl]-N,N,N-trimethylammonium salt (DPTAP),(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium salt (DOTAP).

In a particularly preferred composition, saturated syntheticphosphatidyl cholines such as DMPC, DPPC or DSPC, cholesterol, thecationic lipids DC-Chol, DAC-Chol, DMTAP, DPTAP or DOTAP are used, andin a particularly preferred fashion the proportion of cationic lipids isbetween 5 and 20 mole-% and that of cholesterol between 35 and 50%.

In another advantageous embodiment of the invention, pH-sensitivelycationic lipids are used, as disclosed in WO 02/066490 and U.S. Pat. No.5,965,434 in an exemplary fashion. Liposomes containing such lipids canbe imparted with a state of neutral charge by changing the pH, allowingeasy removal of externally adhering active substance during theproduction process. Examples of pH-sensitively cationic compounds are:

-   histaminylcholesterol hemisuccinate (His-Chol),    morpholine-N-ethylaminocholesterol hemisuccinate (Mo-Chol),    4-(2,3-bis-palmitoyloxy-propyl)-1-methyl-1H-imidazole (DPIM),    cholesterol-(3-imidazol-1-ylpropyl) carbamate (CHIM).

The size of the liposomes according to the invention varies from 20 to1000 nm, preferably from 50 to 800 nm, and more preferably from 50 to300 nm.

Methods established in the prior art, such as extrusion throughpolycarbonate membranes, ethanol injection or high pressurehomogenization, are used to produce the liposomes.

Passive inclusion is preferably used in those cases where large amountsof a readily soluble active substance are to be entrapped. To this end,liposomes with a lipid concentration of from 30 to 150 mM, preferablywith a lipid concentration of from 50 to 120 mM, and more preferablywith a lipid concentration of from 80 to 110 mM are produced in thepresence of dissolved active substance.

Another method of entrapping water-soluble active substances is theso-called “advanced loading” method described in WO 01/34115 A2 whichhereby is incorporated in the disclosure of the present invention. Thismethod enables high inclusion efficiency. It is preferably used in thosecases where the active substance is to be enclosed in the liposomes in apreferably cost-saving manner. This method, which is based on theinteraction between the active substance and membrane-formingsubstances, operates at low ionic strength and at a pH value where theactive substance is present in a state of anionic charge so as toundergo reversible electrostatic interaction with the cationic liposomalmembrane.

For many proteins or peptides, this is the case under physiologicalconditions, i.e., at a pH value between 7 and 8. The charge of theactive substances at a given pH can be inferred from data bases, such asSWISS-PROT, or can be estimated using well-known algorithms.

In another embodiment of the invention the passive inclusion method iscombined with the advanced loading process. In this procedure, theadvanced loading process is performed using a lipid concentration offrom 30 to 150 mM, preferably a lipid concentration of from 50 to 120mM, and more preferably a lipid concentration of from 80 to 110 mM, inorder to significantly increase the inclusion rates compared to theseparate methods.

Following liposome preparation, active substance adhering on the outsideof the liposomal membrane can be detached and removed from the surfaceof the liposomes. This step is of crucial importance to the propertiesof the liposomal depot. Detaching the active substance from the liposomesurface and removing it from the liposome suspension affords depotformulations having virtually no or only minimal “burst release”. Inparticular, this property is of crucial importance in those cases whereactive substances are to be administered which may give rise to toxicreactions in the body even during a briefly high concentration of activesubstance, as is the case during initial arrival. One example for thisis insulin, overdosage of which may give rise to live-threateninghypoglycemic conditions. Termination of the existing interaction can beeffected e.g. by changing the pH value or increasing the ionic strength.

Final removal can be effected using methods well-known to those skilledin the art, such as centrifugation, ultrafiltration, dialysis, or otherchromatographic methods, so that at least 90% of the active substance isentrapped in the liposome and less than 10%, preferably less than 5% ofthe active substance is outside the liposome.

In another embodiment of the invention the active substance adhering tothe liposomal membrane is not detached from the membrane, i.e., the pHvalue or ionic strength remains unchanged. In particular, thisembodiment finds use with active substances where initial arrival of theactive substance is toxicologically safe, as is the case e.g. withleuprolide acetate or many antibodies.

All or part of the free active substance, but more than 5%, preferablymore than 10%, remains in the liposome suspension, providing for rapidinitial arrival of active substance in the blood.

Another advantage of this embodiment is that the suspension can belyophilized because, having equal concentrations of active substance onboth the inner and outer surface of the membrane, release of activesubstance entrapped inside is minimized during the lyophilizationprocess.

Leuprolide acetate ([D-Leu⁶Pro⁹Des-Gly¹⁰]-LHRH ethylamide) is asynthetically produced agonist of LHRH (luteinizing hormone-releasinghormone) and finds clinical use especially in cases of prostate cancer,endometriosis and premature puberty to lower the androgen level in theserum. Continuous administration of leuprolide acetate initially resultsin an increase of the testosterone level which is subsequently lowereddown to the castration level. The initial increase of testosterone isdue to stimulation of the LHRH receptors in the hypophysis and a thusinduced secretion of LH which in turn stimulates testosterone productionin the testicles. Eventually, said initial stimulation by leuprolideacetate is followed by a desensitization of the receptors in thehypophysis, thereby inhibiting the secretion of LH, which results in adecrease of the testosterone level. In a particularly preferredembodiment of the invention, leuprolide acetate is used as activesubstance of a depot system according to the invention.

In another preferred embodiment of the invention, antigens or antigenfragments are used as active substances of an inventive depot system forvaccination. In another preferred embodiment, therapeutically usefulinsulins are employed as active substances in a delivery systemaccording to the invention.

The liposomal formulations of the invention can be used to produce adrug. In a preparatory step the liposomal formulations are placed in aphysiologically tolerable medium. The conditions of a physiologicallytolerable medium are well-known to those skilled in the art, comprisinge.g. a pH value of from 7.3 to 7.6, preferably from 7.4 to 7.5, a saltcontent corresponding to about 150 mM NaCl or an osmolarity of about 320osm.

The liposomal formulations of the invention can be injectedsubcutaneously or intramuscularly as a depot medicinal form.Furthermore, they can also be applied locally or topically.

The invention also relates to a kit comprising the depot systemaccording to the invention, optionally together with informationconcerning combining the contents of the kit. The kit can be used inbasic research and medicine. For example, the information can also be areference to an internet address where further information can beobtained. The information can be a treatment regimen for a disease ore.g. instructions of how to use the kit in research.

Without intending to be limiting, the invention will be explained inmore detail with reference to the following examples.

DESCRIPTION OF THE FIGURES

FIG. 1 Comparison of liposomal depot systems of Example 4 of the presentinvention with an injected control sample (K3) in an animal model.

FIG. 2 Comparison of liposomal depot systems with leuprolide acetate ofExample 4 of the present invention with an injected control sample (P29)in an animal model (serum level of leuprolide acetate).

FIG. 3 Comparison of liposomal depot systems with leuprolide acetate ofExample 4 of the present invention with an injected control sample (P29)in an animal model (serum level of testosterone).

FIG. 4 Liposomal depot system with leuprolide acetate of Example 7 ofthe present invention in an animal model (serum level of leuprolideacetate)

EXAMPLES Example 1 Inclusion of Insulin in Liposomes

Lipid mixtures having the following composition Formulation CompositionI-1 DPPC/DC-Chol/Chol 60:10:30 (mole-%) I-2 DPPC/DOTAP/Chol 50:10:40(mole-%)are dissolved in chloroform at 50° C. and subsequently dried completelyin vacuum in a rotary evaporator. The lipid film is added with humaninsulin solution (recombinant insulin; 4 mg/ml insulin in 10 mM HEPES,300 mM sucrose, pH 7.5) in an amount so as to form a 50 mM suspension.Subsequently, this suspension is hydrated in a water bath at 50° C. for45 minutes by agitating and treated in an ultrasonic bath for another 5minutes. Thereafter, the suspension is frozen. This is followed by 3cycles of freezing and thawing, each thawing being followed by a 5minute treatment in the ultrasonic bath.

Following final thawing, the liposomes are subjected to multipleextrusions through a membrane having a pore width of 200 nm or 400 nm(Avestin LiposoFast, polycarbonate membrane with a pore width of 200 or400 nm). Following extrusion, the resulting suspension is rebuffered byadding a stock solution of glycine-HCl, pH 3.5, and NaCl. Afterfiltration of the liposomes through 0.8 μm, non-entrapped insulin isremoved by triple sedimentation in an ultracentrifuge at 60,000×g, 45min. A physiological pH is readjusted by adding a HEPES stock solution,pH 7.5. The amount of entrapped insulin is determined followingextraction with CHCl₃ and CH₃OH, using RP-HPLC. Inclusion rates of80-100% insulin are found.

Example 2 Inclusion of Alkaline Phosphatase (AP) in Liposomes

A lipid mixture having the following composition Formulation CompositionAP-1 DPPC/DOTAP/Chol 50:10:40 (mole-%)is dissolved in chloroform at 50° C. and subsequently dried completelyin vacuum in a rotary evaporator. The lipid film is added with APsolution (from bovine intestinal mucosa) (5 mg/ml AP in 10 mM HEPES, 300mM Sucrose, pH 7.5) in an amount so as to form a 50 mM suspension.Subsequently, this suspension is hydrated in a water bath at 50° C. for45 minutes by agitating and treated in an ultrasonic bath for another 5minutes. Thereafter, the suspension is frozen. This is followed by 3cycles of freezing and thawing, each thawing being followed by a 5minute treatment in the ultrasonic bath.

Following final thawing, the liposomes are subjected to multipleextrusions through a membrane having a pore width of 200 nm or 400 nm(Avestin LiposoFast, polycarbonate membrane with a pore width of 200 or400 nm). Following extrusion, the ionic strength of the resultingsuspension is increased by adding a stock solution of NaCl.

Removal of non-entrapped AP is effected by triple sedimentation in anultracentrifuge at 60,000×g for 45 min.

Following organic precipitation with CHCl₃ and CH₃OH, the amount ofentrapped AP is determined using a protein assay (BCA Protein AssayReagent Kit, Perbio). In addition, the activity of entrapped AP isdetermined using an enzyme assay (p-nitrophenylphosphate test) .Inclusion rates of 40-50% AP are found.

Example 3 Inclusion of Inulin in Liposomes

Lipid mixtures having the following composition Formulation CompositionP-20 DPPC/DC-Chol/Chol 60:10:30 (mole-%) P-21 DPPC/DOTAP/Chol 50:10:40(mole-%) P-23 DPPC/DPPG 40:60 (mole-%)are dissolved in chloroform at 50° C. and subsequently dried completelyin vacuum in a rotary evaporator. The lipid film is added with ³H-inulinsolution (18.5 MBq/ml ³H-inulin in 10 mM HEPES, 150 mM NaCl, pH 7.5) inan amount so as to form a 100 mM suspension. Subsequently, thissuspension is hydrated in a water bath at 500C for 45 minutes byagitating. Thereafter, the suspension is frozen. This is followed by 3additional cycles of freezing and thawing.

After the third thawing, the liposomes are subjected to multipleextrusions through a membrane having a pore width of 200 nm (AvestinLiposoFast, polycarbonate membrane with a pore width of 200). Removal ofnon-entrapped ³H-inulin is effected via gel filtration (G75 columnPharmacia). Following removal, the amount of entrapped ³H-inulin isdetermined in a scintillation counter. Inclusion rates of 10-25%³H-inulin are found.

Example 4 Use of Liposomal Depot Systems in an Animal Model

The different liposomes of Example 3 were injected subcutaneously inhealthy rats (3 animals per group) at a concentration of 20 mM lipid ina volume of 0.5 ml. A control sample with blank liposomes andnon-encapsulated ³H-inulin was likewise administered subcutaneously in avolume of 0.5 ml. The pharmacokinetic data was obtained by bloodsampling at varying points in time. The test period of the animal studywas 6 weeks in total. The general condition of all animals was good overthe test period. Only one animal in Group P20 showed heavy breath soundsfor about 1 hour on test day 10.

The inulin content was determined by combustion of the blood samples(Oxidizer Ox 500, Zinser) and subsequent scintillation measurements.

The formulations and relative bioavailabilities up to t=42 d areillustrated in the following table: Relative bioavailability up to t =Formulation Composition 42 days [%] K-3 DPPC/DPPG/Chol 50:10:40 (200nm) + 100 ³H-inulin outside P-20 DPPC/DC Chol/Chol 60:10:30 (200 nm)136.5 P-21 DPPC/DOTAP/Chol 50:10:40 (200 nm) 120 P-23 DPPC/DPPG 40:60(200 nm) 142

Example 5 Inclusion of Leuprolide Acetate in Liposomes

Lipid mixtures having the following composition Formulation CompositionP-26 DPPC/DC-Chol/Chol 60:10:30 (mole-%) P-27 DPPC/DOTAP/Chol 50:10:40(mole-%) L1 DPPC/DC-Chol/Chol 60:10:30 (mole-%) (no removal)are dissolved in chloroform at 50° C. and subsequently dried completelyin vacuum in a rotary evaporator. The lipid film is added withleuprolide acetate solution (95 mg/ml in 10 mM HEPES, 150 mM NaCl, pH 6,L1: 2.5 mg/ml) in an amount so as to form a 100 mM suspension.Subsequently, this suspension is hydrated in a water bath at 50° C. for45 minutes by agitating. Thereafter, the suspension is frozen. This isfollowed by 3 additional cycles of freezing and thawing. Following finalthawing, the liposomes are subjected to multiple extrusions through amembrane having a pore width of 400 nm (Avestin LiposoFast,polycarbonate membrane with a pore width of 400 nm). Removal ofnon-entrapped leuprolide acetate is effected by means of triplesedimentation in an ultracentrifuge at 60,000×g for 45 minutes (not withL1). The amount of entrapped leuprolide acetate is determined followingextraction with CHCl₃ and CH₃OH, using RP-HPLC. Inclusion rates of about15% leuprolide acetate are found.

Example 6 Use of Liposomal Depot Systems in an Animal Model

The different liposomes of Example 5 were injected subcutaneously inhealthy male rats (3 animals per group) at a concentration of 25-30 mMlipid in a volume of 0.5 ml. A control sample with blank liposomes andnon-encapsulated leuprolide acetate was likewise administeredsubcutaneously in a volume of 0.5 ml. The pharmacokinetic data wasobtained by blood sampling at varying points in time, obtaining serumand determining the leuprolide acetate concentration in the serum bymeans of ELISA (Peninsula).

As leuprolide acetate influences the testosterone level of male rats,the testosterone concentration in the serum was also determined over theentire period using ELISA (DRG)

The test period of the animal study was 6 weeks in total. The generalcondition of all animals was good over the test period. The formulationsand relative bioavailabilities up to t=42 d are illustrated in thefollowing table: Relative bioavailability Size up to t = FormulationComposition [nm] 42 days [%] P-29 DPPC/DC-Chol/Chol 60:10:30 + 290 100leuprolide, outside P-26 DPPC/DC-Chol/Chol 60:10:30 305 117

Example 7 Use of Liposomal Leuprolide Acetate in an Animal Model

Without removal of the active substance present outside, the liposomesof Example 5 were injected subcutaneously in healthy male rats (3animals per group) in a volume of 0.5 ml. The leuprolide acetate dosewas 2.5 mg per animal. The pharmacokinetic data was obtained by bloodsampling at varying points in time, obtaining serum and determining theleuprolide acetate concentration in the serum by means of ELISA(Peninsula). The test period of the animal study was 6 weeks in total.The general condition of all animals was good over the test period. Theformulation is shown in the following table: Dose FormulationComposition [mg] L1 DPPC/DC-Chol/Chol 60:10:30 no removal 2.5

Example 8 Inclusion of Cy5.5 Anti-CD40 ODN (Antisense Oligonucleotide)in Liposomes

A lipid mixture having the following composition: FormulationComposition AS1 DPPC/DC-Chol/Chol 60:10:30 (mole-%)is dissolved in chloroform at 50° C. and subsequently dried completelyin vacuum in a rotary evaporator. The lipid film is added with Cy5.5anti-CD40 ODN (antisense oligonucleotide; 150 μg/ml in 10 mM HEPES, 300mM Sucrose, pH 7.5) in an amount so as to form a 15 mM suspension.Subsequently, this suspension is hydrated in a water bath at 50° C. for45 minutes by agitating and treated in an ultrasonic bath for another 5minutes. Thereafter, the suspension is frozen. This is followed by 3cycles of freezing and thawing, each thawing being followed by a 5minute treatment in the ultrasonic bath.

Following final thawing, the liposomes are subjected to multipleextrusions through a membrane having a pore width of 200 nm or 400 nm(Avestin LiposoFast, polycarbonate membrane with a pore width of 200 or400 nm). Following extrusion, the ionic strength of the resultingsuspension is increased by adding a stock solution of NaCl.

Following removal of free active substance by triple sedimentation in anultracentrifuge at 60,000×g for 45 min, the amount of entrapped Cy5.5anti-CD40 ODN (antisense oligonucleotide) is determined usingfluorescence spectroscopy.

The inclusion efficiency of the oligonucleotides is around 47%.

1. A depot system, for delayed release of active substances comprisingliposomes having a membrane the liposomes comprising saturated syntheticphosphatidyl cholines selected from one or more from the groupconsisting of DMPC, DPPC and DSPC, cholesterol with a percentage rangingfrom 35 to 50 mole-%, cationic lipids selected from the group ofDC-Chol, DAC-Chol, DMTAP, DPTAP and DOTAP with a percentage ranging from5 to 20 mole-% in the liposomal membrane, and one or more selected fromthe group consisting of protein and peptide active substances.
 2. Thedepot system according to claim 1, wherein the cationic lipids arecationic in a pH-sensitive fashion and selected from one or more fromthe group consisting of His-Chol and Mo-Chol.
 3. The depot systemaccording claim 1, wherein at least 90% of the active substance isenclosed in the liposome and less than 10% is outside the liposome. 4.The depot system according to claim 1, wherein the active substance isentrapped in the liposome and more than 10% thereof is outside theliposome.
 5. The depot system according to claim 1, wherein the depotsystem is capable of sustaining the delivery of the active substance forat least 1 week.
 6. The depot system according to claim 1, wherein thesize of the liposomes varies from 20 to I ,000 nm. 7-18. (canceled) 19.The depot system according to claim 1, wherein the size of the liposomesvaries from 50 to 800 nm.
 20. The depot system according to claim 1,wherein the size of the liposomes varies from 50 to 300 nm.
 21. Thedepot system according to claim 1, wherein the active substancecomprises one or more from the group consisting of LHRH agonists andGnRH analogs.
 22. The depot system according to claim 21, wherein theactive substance comprises one or more from the group consisting ofleuprolide, acetate, buserelin, goserelin and triptorelin.
 23. The depotsystem according to claim 1, wherein the said active substance comprisesinsulin.
 24. The depot system according to claim 1, wherein the saidactive substance comprises heparin.
 25. The depot system according toclaim 1, wherein said active substance comprises antigen fragments forvaccination.
 26. The depot system according to claim 1, wherein thedepot system is capable of a delayed release of active substance for atleast one week and said active substance comprises oligonucleotides. 27.The depot system according to claim 26, wherein said oligonucleotidesare constituted of 5-100 from the group consisting ofdeoxyribonucleotides, ribonucleotides and chemically modifiedderivatives thereof.
 28. The depot system according to claim 26, whereinsaid oligonucleotides are constituted of 5-40 from the group consistingof deoxyribonucleotides, ribonucleotides and chemically modifiedderivatives thereof.
 29. The depot system according to claim 26, whereinsaid oligonucleotides are constituted of 10-25 from the group consistingof deoxyribonucleotides, ribonucleotides and chemically modifiedderivatives thereof.
 30. The depot system according to claim 26, whereinsaid oligonucleotides are present as one or more from the groupconsisting of a single strand, a double strand and in complex folding.31. The depot system according to claim 30, wherein saidoligonucleotides are present as a single strand, said single strandbeing present as antisense oligonucleotides.
 32. The depot systemaccording to claim 30, wherein said oligonucleotides are present as adouble strand, said double strand being present as small interferingRNA, decoy oligonucleotides.
 33. The depot system according to claim 30,wherein said oligonucleotides are present in complex folding asaptamers, spieglemers.
 34. The depot system according to claim 1,wherein the depot system is capable of a delayed release of activesubstance for at least one week and said active substance comprises awater-soluble active substance derivative selected from one or more fromthe group consisting of active substances of antibiotic, antimycotic,cytostatic agents and glucocorticoids.
 35. Method of administering thedepot system according to claim 1, comprising the step of injecting thedepot system subcutaneously or intramuscularly.
 36. Method ofadministering the depot system according to claim 1, comprising the stepof one or both from the group consisting of topical and localapplication to support healing processes.
 37. A Drug comprising a depotsystem according to claim 1.